PROJECTSUMMARY(DESCRIPTION) Mutationisthesourceofgeneticvariationandcangiverisetodisease,aging,andcancer.Mammalshaveso- phisticatedpathwaystoreducemutationbyrepairingerrorsoccurringduringDNAreplicationaswellasdam- ageduetoenvironmentaleffects.Whilemanybiologicalpathwayshavebeenextensivelystudied,otherun- knownDNArepairpathwaysmayremainundiscovered.Wehypothesizethatonesuchpathwayisthecircadi- anclockpathway.Thescientificpremiseofourproposalisthatstudieshaveshownmiceharboringgenetic disruptionofcircadianclockfunctionexhibitincreasedtumorformationandacceleratedaging.Further,epide- miologicalstudiessuggestcircadiandisruptionisalikelycarcinogen.Whilemammaliancorecircadianclock proteinsCRY1andCRY2seemtolackcatalyticDNArepairactivity,theyevolvedfrombacteriallight-activated DNArepairenzymes(CPDphotolyases).Lastly,wehaverecentlyshownthatCRY2-deficientcellscontain significantlyelevatednumbersofdoublestrandbreaks,andCry2-/-miceareuniquelybornatsub-Mendelian ratios,suggestinganincreaseinlethalmutations. Basedontheseobservationsandthispremise,wehypothesizethatmammalianCRY2hasmaintainedafunc- tionalconnectiontoprotectinggenomeintegrity,andthatdisruptionofthisfunctionincreasesgenome-wide mutationrates,whichinturncontributestoelevatedcancerriskcausedbyenvironmentalcircadiandisruption. Totestthishypothesis,wewillmeasuremutationratesinwildtypeandCry2-/-littermatemicesubjectedto standardhousingconditionsortoenvironmentalcircadiandisruptionbyperformingwhole-genomesequencing. InAim1ofthisproject,wewilltestwhetherCry2-/-micehaveelevatedgermlinemutationratescomparedwild- type.Todothis,wewillperformthreeindependentcrossesfromwhichwewillsequencebothparentsand threeoftheirprogenyandcountthenumberofdenovomutationspresentintheprogeny.Wewillcomparethe numberofaccumulatedmutationsinprogenyborntoCry-deficientparentstothatinoffspringborntowild-type parents.Becausemutationsmaynotoccurequallyinmaleandfemaleparents,wewillexamineoffspringfrom maleorfemaleCry2-deficientparentsbredtowildtypepartners.InAim2ofthisproject,wewilltestwhether environmentalcircadiandisruptionincreasesgermlinemutationrates.Todothis,wewillbreedamouseex- posedtochronicjetlaglightcyclestoawild-typeparentandsequencethegenomesoftheparentsand3of theprogeny.Wewillcomparethenumberofdenovomutationstothatnumberinoffspringborntoparentsex- posedtonormallight-darkcycles.Successfulcompletionofthisresearchwillindicatewhetherenvironmental disruptionofcircadianclockscanaffectgenomeintegrity,layingthegroundworkforfurtherstudiesofthe mechanismofcircadiandisruptionondisease.